Differentiation of human pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-9752126-B2
Application number	US-201514963436-A
Country	US
Kind code	B2
Filing date	Dec 9, 2015
Priority date	Oct 31, 2008
Publication date	Sep 5, 2017
Grant date	Sep 5, 2017

Abstract

The present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependent kinase inhibitor to cause an increase in expression of MAFA.

First claim

What is claimed is:

1. A method for increasing the expression of insulin and MAF bZIP transcription factor A (“MAFA”) in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of:
a. sequentially differentiating human pluripotent stem cells to obtain cells expressing markers characteristic of the pancreatic endocrine lineage; and
b. culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising an added amount of a cyclin-dependent kinase inhibitor to cause an increase in expression of insulin and MAFA as compared to cells expressing markers characteristic of the pancreatic endocrine lineage that are not cultured in medium comprising the added cyclin-dependent kinase inhibitor;
wherein the cyclin-dependent kinase inhibitor is selected from group consisting of 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide and 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

2. The method of claim 1 , wherein the cyclin-dependent kinase inhibitor is added to cells expressing markers characteristic of the endocrine lineage at a concentration from about 0.1 μM to about 10 μM for about one to seven days.

3. The method of claim 1 , wherein the cells expressing markers characteristic of the pancreatic endocrine lineage are pancreatic endocrine cells.

4. The method of claim 1 , wherein the cyclin-dependent kinase inhibitor is 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide.

5. The method of claim 1 , wherein the cyclin-dependent kinase inhibitor is 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

6. A method for increasing the expression of insulin and MAF bZIP transcription factor A (“MAFA”) in a population of cells comprising pancreatic endocrine cells comprising culturing the population of cells in medium comprising an added amount of a cyclin-dependent kinase inhibitor to cause an increase in the expression of insulin and MAFA in the population as compared to a population of cells comprising pancreatic endocrine cells not cultured in medium comprising the added cyclin-dependent kinase inhibitor, wherein the cyclin-dependent kinase inhibitor is selected from group consisting of 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide and 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

7. The method of claim 6 , wherein the method comprises adding the cyclin-dependent kinase inhibitor at a concentration from about 0.1 μM to about 10 μM for about one to seven days.

8. The method of claim 6 , wherein the cyclin-dependent kinase inhibitor is 5-amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1h-1,2,4-triazole-1-carbothioamide.

9. The method of claim 6 , wherein the cyclin-dependent kinase inhibitor is 2-bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione.

10. The method of claim 6 , wherein the pancreatic endocrine cells are obtained by differentiating human pluripotent stem cells.

11. The method of claim 10 , wherein the human pluripotent stem cells are human embryonic cells.

Assignees

• Janssen Biotech Inc

Inventors

• Rezania Alireza
• Fryer Benjamin

Classifications

• C12N2501/155

  Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N2501/998

  Proteins not provided for elsewhere · CPC title

• C12N2501/41

  Hedgehog proteins; Cyclopamine (inhibitor) · CPC title

• C12N5/0676 Primary

  Pancreatic cells · CPC title